7-{8 3-(Phenyl)-isoxazol-5-yl{9 acetamido-cephalosporanic acids

ABSTRACT

Novel heterocyclic amides of the formula   WHEREIN R is selected from the group consisting of lower alkyl and aryl optionally substituted with at least one member of the group consisting of chlorine, fluorine, nitro, amino and lower alkyl and a tertiary alkyl group, R1 is selected from the group consisting of hydrogen, lower alkyl, carboxyl, lower alkoxycarbonyl, an alkali metal, alkaline earth metal or amine salt of carboxyl, a carbamyl, cyano, an amino and chlorine, R2 is selected from the group consisting of hydrogen, cyano, halogen, an amino, lower aralkoxycarbonylamino, lower alkyl, carboxyl esterified with lower alkyl, aryl or aralkyl and carbamoyl optionally substituted on the nitrogen atom with lower alkyl or a phenyl and Q is selected from the group consisting of   X is selected from the group consisting of hydrogen, hydroxy and lower alkanoyloxy and U is selected from the group consisting of amido forming groups or a group OY, wherein Y is selected from the group consisting of hydrogen, salt forming groups, and ester forming groups, or COOY and CH2X together form a lactone or lactam, having antibacterial properties, their preparation and novel intermediates thereof.

United States Patent 1 Henniger et al.

[22] Filed: Nov. 3, 1971 [2l] Appl. No.: 195,482

[30] Foreign Application Priority Data Nov. 6, l970 United Kingdom 53040/70 [52] US. Cl 260/243 C; 260/239.l; 424/246 [51 Int. Cl C07d 99/24 [58] Field of Search 260/243 C, 239.1

[56] References Cited UNITED STATES PATENTS 3,218,318 11/1965 Flynn 260/243 C 3,459,746 8/1969 Flynn 260/243 C Primary Examiner-Donald G. Daus Assistant Examiner.lose Tovar Attorney, Agent, or FirmHammond 8L Littell [57] ABSTRACT Novel heterocyclic amides of the formula June 24, 1975 wherein R is selected from the group consisting of lower alkyl and aryl optionally substituted with at least one member of the group consisting of chlorine, fluorine, nitro, arhino and lower alkyl and a tertiary alkyl group, R, is selected from the group consisting of hydrogen, lower alkyl, carboxyl, lower alkoxycarbonyl, an alkali metal, alkaline earth metal or amine salt of carboxyl, a carbamyl, cyano, an amino and chlorine, R is selected from the group consisting of hydrogen, cyano, halogen, an amino, lower aralkoxycarbonylamino, lower alkyl, carboxyl esterified with lower alkyl, aryl or aralkyl and carbamoyl optionally substituted on the nitrogen atom with lower alkyl or a phenyl and Q is selected from the group consisting of X is selected from the group consisting of hydrogen, hydroxy and lower alkanoyloxy and U is selected from the group consisting of amido forming groups or a group OY, wherein Y is selected from the group consisting of hydrogen, salt forming groups, and ester forming groups, or COOY and CH X together form a lactone or lactam, having antibacterial properties, their preparation and novel intermediates thereof.

10 Claims, No Drawings 1 7-[3-(PHENYL)[SOXAZOL-S-YL]ACETAMIDO- CEPHALOSPORANIC ACIDS OBJECTS OF THE INVENTION THE INVENTION The novel heterocyclic compositions of the invention have the formula wherein R is selected from the group consisting of lower alkyl and aryl optionally substituted with at least one member of the group consisting of chlorine, fluorine, nitro, amino and lower alkyl and a tertiary alkyl group, R. is selected from the group consisting of hydrogen, lower alkyl, carboxyl, lower alkoxycarbonyl, 3

an alkali metal, alkaline earth metal or amine salt of carboxyl. a carbamyl, cyano, an amino and chlorine, R is selected from the group consisting of hydrogen, cy-

E? l C H AT-- ano, halogen, an amino, lower aralkoxycarbonylamino, lower alkyl, carboxyl esterified with lower alkyl, aryl or aralkyl and carbamoyl optionally substituted on the nitrogen atom with lower alkyl or a phenyl and O is selected from the group consisting of X is selected from the group consisting of hydrogen, hydroxy and lower alkanoyloxy and U is selected from the group consisting of amido forming groups or a group OY wherein Y is selected from the group consisting of hydrogen, salt forming groups, ester forming groups, or COOY and CH X together form a lactone or lactam. The term lower" is intended to mean I to 4 carbon atoms.

III

Among the preferred substituents of formula I, R is lower alkyl, phenyl or naphthyl optionally substituted with one or more of chlorine, fluorine, nitro, amino or lower alkyl, preferably 2,6-dichlorophenyl or tertiary alkyl such as adamantyl, R. is hydrogen, lower alkyl, COOY where Y is hydrogen, lower alkyl, alkali metal, alkaline earth metal or an amino group, carbamoyl, cyano, amino or chlorine and R is hydrogen. CN, amino, lower aralkoxycarbonylamino, lower alkyl, lower alkoxycarbonyl, phenyl, lower aralkyl such as benzyl and carbamoyl optionally substituted on the nitrogen atom with lower alkyl or phenyl, X may be hydrogen, OH or lower alkanoyloxy such as acetoxy, or -CI-l X together with the -COOH group may form and U is an amide forming group such as -NI-l saccharinyl, succinimido, phthalimido or a group OY. wherein Y is hydrogen, alkali metal, alkaline earth metal or amine salts, an ester group such as tri(lower) alkylsilyl, di(lower)alkylmonohalosilyl, benzyl, phenacyl or lower alkyl.

The term lower" as applied to alkyl or alkanoyl is intended to mean 1 to 4 carbon atoms.

The compounds of formula I may be prepared by several different methods, each of which is an application of a method known in the art for the preparation of penicillins and cephalosporins. According to a feature of the invention. the compounds of formula I are prepared by reacting a salt, ester of a fi-aminopenicillanic or 7-aminocephalosporanic acid compound of the formula:-

'1 13 (wherein X is as defined above) with the substituent X when a hydroxy group preferably protected, with an active ester (e.g. 2,4-dinitrophenyl ester, p-nitrophenyl ester of N-hydroxysuccinimide ester) with an acid of the formula:-

wherein R, R, HHCLR: are as defined above or an active functional derivative thereof suitable as an acylating agent for a primary amino group. Such derivatives include the corresponding carboxylic acid chlorides, bromides, acid anhydrides, including mixed anhydrides.

prepared from stronger acids such as lower aliphatic monoesters of carbonic acid, or alkyl and aryl sulfonic acids and of more sterically hindered acids such as diphenylacetic acid. Moreover, an acid azide or active 3 {p 9% n-cooa O=C=N-CH on C A-CO-NH-CH on ters, a corresponding azolide, i.e. an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five membered ring containing at least two nitrogenatoms i.e., imidazole, pyrazole, triazoles,

benzimidazole, benzotriazole and their substituted derivatives can be used.

The methods for carrying out these reactions to produce a penicillin or a cephalosporin and the methods used to isolate the compounds so produced are well known in the art such as the British Patents Nos. 932,644, 957,570, 959,054, 952,519, 932,530. 967,108 and 967,890.

The ester, salt or amide of the product obtained by the aforesaid processes may be converted by methods known per se into the corresponding penicillanic or cephalosporanic acids. For example, when a silyl (e.g. trialkylsilyl) ester of the starting material of formula IV or V is employed as reactant, the esterifying group can be readily hydrolyzed to yield the corresponding acid compound of formula 1.

Another method of the invention for preparing the compounds of general formula 1 comprises reacting an acid of the formula A-COOH wherein A is the group:-

VII

in which R, R, and R are as defined above) having reactive groups in the radical A suitably protected. with a 6-isocyanatopenicillanic acid or 7-isocyanato- (desacetoxy)cephalosporanic acid compound of the formula =C=NQ (wherein Q is as defined above) having atoms or groups protecting the carboxyl group and optional hydroxy group, when present (i.e. when X in formula lll is hydroxyl). Preferably the group protecting the carboxyl radical or hydroxy radical when present in the 6-isocyanatopenicillanic or 7-isocyanatocephalosporanic reactant is a dior trialkylsilyl group which can readily be removed from the resultant product by hydrolysis.

The reac tion between the carboxylic acid of formula A-COOH and isocyanate of formula OCNQ is preferably carried out in an inert organic solvent medium such as toluene, dichloromethane or benzonitrile. A small amount of an organic base, for example a substituted imidazole, may serve as catalyst. The reaction proceeds according to the reaction scheme illustrated below for penicillanic acid derivatives, for example with a protecting ester group:

CH3 C0 H N CH COOE COOE wherein E is a group protecting the carboxyl group during the reaction and is removed, for example by hydrolysis, after the reaction.

in another method for preparing the penicillanic and cephalosporanic acid derivatives of formula I, a 6- isocyanatopenicillanic or 7-isocyanatocephalosporanic acid compound O=C=N-Q wherein Q is as defined above having the carboxyl group, and hydroxy group when present, suitably protected, is reacted with an organo-metal compound of the formula A-Me'. A-Me" Hal or A-Me"A wherein A is as defined above, Me is a metal atom such as lithium, sodium or magnesium, the numeral l or ll indicating its valency, and Hal is a halogen (preferably chlorine or bromine) atom followed by hydrolyzing the intermediate product obtained to remove the metal ion, and any hydrolyzable group protecting the carboxyl group. The reaction is carried out in an anhydrous organic solvent medium under conditions favoring a reaction of the Grignard, Reformatsky or analogous type.

The isocyanate starting materials of the formula O=C=N-Q wherein Q is as defined above can be prepared by reacting phosgene with a penicillanic or cephalosporanic acid derivative of the formula wherein W is hydrogen or a group such that the group W-NH- is easily convertible into an isocyanato group by reaction with phosgene, and the group O is as defined above with the carboxyl, and hydroxy group when present, suitably protected. The group W in the starting material may be introduced on the amino group of the o-aminopenicillanic acid or 7-aminocephalosporanic acid derivative concurrently with the protection of the carboxyl group and hydroxyl group or afterwards. Preferably, W is a tri(lower) alkylsilyl group. When W is an easily removable group, the reaction of such compounds with phosgene proceeds much more smoothly under the same reaction conditions than is the case when W is a hydrogen atom. The reaction with phosgene'must be carried out in a dry. inert organic solvent medium having regard to the reactivity of the resulting isocyanato groups. Toluene and methylene chloride or mixtures thereof are particularly suitable.

To facilitate the reaction. an organic base can be added to bind the hydrogen chloride formed. Preferably this base is a tertiary amine such as triethylamine which does not react with the isocyanato function. As high temperatures would lead to decomposition of the penicillanic acid or cephalosporanic acid nucleus, the reaction is preferably carried out at very low temperatures. preferably -40C.

The substituted isoxazol-S-yl acetic acid starting ma- The new penicillanic and cephalosporanic acid derivatives of formula I have antibiotic properties which make them useful with human beings and animals alone or mixed with other known antibiotics. Some of the terials of formula VI, most of which are new and thus 5 new compounds of formula l have activities comparaform an additional feature of the invention, were prepared by the following processes:

route 1 N CH 9 R-C N O route 2 The starting nitrile oxides can be prepared by known methods, as described by Grundmann, Quilico et al., See e.g. Synthesis 1970, 344 and Experienta 26, H6) (1970) and references cited herein. The reaction with n-butyllithium in the presence of tetramethyleendiamine (TMEDA) can be carried out in aprotic solvents such as toluene or tetrahydrofuran. Route 1 enables the greatest variation in the desired compounds. In some cases. the group R1 has been changed into another group. e.g. COOH into CONH or CN or NH- after the l,3 -dipolar addition reaction, but before the lithiation in order to avoid the synthesis of the starting acetylenes which are sometimes difficult to prepare. The introduction ofa group R H in formula Vli can be carried out directly via route 1 by starting with lbutyne (R =CH instead of propyne.

The other processes comprise l) a-halogenation of compounds of formula VI optionally followed by reaction with a nucleophilic agent. e.g. oz-amino acid (formula Vl R=2.6-dichlorophenyl. R =H, R =NH- was prepared via a-bromination of the corresponding methyl isoxazol-S-yl-acetate with l,3-dibromo-5,5- dimethylhydantoin followed by hydrolysis and reaction of the a-bromo acid with concentrated ammonia. This was an improved synthesis analogus to that one of ibotenic acid as described in Chem. Pharm. Bull. 14, 89 1966) and 2) via a-lithiation of the carboxylic acids of formula VI, and reaction with an appropriate agent.

Examples of new compounds of formula VI which can be prepared by the depicted scheme are those EMJ -CH CH OH ble to those of penicillin G and they have special activities against gram positive organism and have. more- Buli/TMEDA a; CO2 H3O R I.) i li -ca COOI-I oxidation over, a good activity against penicillin resistant Staphy- 25 lococci, especially the compounds in which R repre sents the 2,6-dichlorophenyl group. R, is hydrogen or methyl. R is hydrogen and Q is the group of formula II or III in which X represents an acetoxy group, and salts of such compounds.

The compounds according to the invention are preferably employed for therapeutic purposes in the form of a non-toxic pharmaceutically acceptable salt such as the sodium, potassium or calcium salt. Other salts that may be used include the non-toxic, suitably crystaline salts with organic bases such as amines, for example tri(lower)alkylamines, procaine and dibenzylamine.

The novel antibacterial compositions of the invention are comprised of a bactericidally effective amount of a 40 compound of formula I and a pharmaceutical carrier.

The said compositions may be in the form of liquid preparation such as solutions, suspensions, dispersions or emulsions or in solid form such as powders, capsules or tablets. One or more other therapeutics may be 45 added to the said compositions.

The term effective amount" as used herein in relation to the described compounds means an amount which is sufficient to destroy or inhibit the growth of susceptible microorganism when administered in the usual manner or an amount which is sufficient to control the growth of bacteria. The magnitude of an effective amount can be easily determined by those in the art through standard procedures for determining the relative activity of antibacterial agents, when utilized against susceptible organisms via the various available routs of administration.

Suitable carriers and excipients may be any convenient physiologically acceptable ingredient which can serve to facilitate administration of the therapeutically active compound. Carriers may provide some ancillary function such as that of a diluent, flavor-masking agent, binding agent. action delaying agent, stabilizer, and the like. Illustrative carriers include water which can contain gelatin, acacia. algenate, dextran, polyvinylpyrrolidine, sodium carboxymethyl cellulose. or the like, aqueous ethanol, syrup, isotonic saline. isotonic glucose, starch, lactose, or any other such material commonly used in the pharmaceutical and veterinary industry.

The novel method of killing bacteria comprises contacting bacteria with a bactericidal amount of a compound of formula 1. When administered to warmblooded animals, the compounds may be administered for example topically or parenterally. The usual daily dose is to 100 mg/kg depending upon the method of administration and the specific compound.

In the following examples these are described several preferred embodiments to illustrate the invention. However, it should be understood that the invention is not intended to be limited to the specific embodiments.

EXAMPLE I "do v In a three-necked flask equipped with a gas inlet tube, thermometer and dropping funnel, 755 mg (3.5 mmoles) of o-aminopenicillanic acid were suspended in 10 ml of ethyl acetate under an atmosphere of nitrogen and the flask was cooled in an ice bath while 0.51 ml (3.8 mmoles) of triethylamine were added followed after 10 minutes by 0.48 ml (3.8 mmoles) of trimethylchlorosilane. Stirring was continued for about minutes and then another 0.51 ml (3.8 mmoles) of triethylamine were added followed by 3-(2,6-dich1orophenyl) isoxazol-S-ylacetyl chloride (prepared by the reaction of thionyl chloride with 3-(2,6- dichlorophenyl)isoxazol-S-ylacetic acid in diethyl ether with a trace of dimethylformamide) in 5 ml of ethyl acetate added drop-wise to the reaction mixture at such a rate that the temperature did not rise above 5C. After the addition, the ice bath was removed and stir ring was continued for another 90 minutes at room temperature.

The reaction mixture was then poured into a mixture of 20 ml of water and 20 ml of diethyl ether with icecooling, the pH being maintained at 6.8. The aqueous layer was washed again with 30 ml of diethyl ether and the aqueous layer was acidified to pH 1.5 after addition of ml of diethyl ether. After separation, the aqueous layer was washed again with 30 ml of diethyl ether and the combined organic layers were washed once with 20 ml of acidified ice-water at pH 1.5 and then with 20 ml of ice-water. After drying and treatment with Norit, the organic layer was concentrated to about half its volume and then sodium oz-ethyl-capronate was added. The precipitated sodium salt is filtered off, washed with diethyl either and dried to obtain 550 mg (32% yield) of the sodium salt of 6-1(3-[2,6-dichlorophenyl] isoxazol- 5-y1) acetamido]-penicil1anic acid. According to TLC the compound was pure.

EXAMPLE ll Sodium salt of 6- 3-( 2 ,6-dich1orophenyl )isoxazole-S-yl]acetamido) penicillanic acid A three-necked vessel of 250 ml was equipped with a thermometer, a good condenser and dropping funnel and the reaction was carried out under nitrogen. 220 ml of dichloromethane and 2.72 g 10 mmol) of 3-(2,6- dichlorophenyl)isoxazol-S-ylacetic acid were introduced into the vessel. After the introduction of 0.13 ml of N-vinylimidazole (a catalyst), a solution of 3.14 g (10 mmol) of trimethylsilyl 6-isocyanatopenicillanate in dichloromethane was added drop-wise to the stirred solution at 20C. After 23 hours, the reaction was complete and the isocyanate was converted to the extent of about into the desired product. The reaction mixture was poured into ice-water buffered to pH 7 and was extracted twice with diethyl ether. The aqueous layer was acidified to pH 4.0 and was extracted three times with diethyl ether. The desired product was completely removed from the aqueous layer and the col- COONa lected organic layers were washed with a small amount of ice-water and then dried over anhydrous magnesium sulfate, filtered and concentrated to some extent in vacuo at 0C.

A solution of sodium a-ethylcapronate in ethyl acetate was added drop-wise to the concentrated solution and the resulting colorless precipitate was collected on a filter, washed with diethyl ether and dried in vacuo to obtain 2.81 g (57%) of the sodium salt of 6-{[3-(2,6- dichlorophenyl )isoxazole-S-yl acetamido l -penicillan ic acid. Analysis of the PMR spectrum of the product dissolved in hexadeuterodimethylsulphoxide (60 Mc,5- values in ppm, internal reference tetramethylsilane):

NH multiplet at 8.95 (0.7 protons) C H about 7.5 (3 protons) isoxazolyl-H 6.50 (proton) 3.99 (3 protons) doublet between 5.50 and 5.30

(2 protons) C Cl stretching vibration 755 9 EXAMPLE m Sodium salt of 7- l 3-( 2.6-dichlorophenyl )-isoxazol-- \'l lacetamido cephalosporanic acid COONa funnel. in IQ ml of ethyl acetate under an atmosphere of nitrogen. The suspension was cooled in an ice-bath and 0.3 ml (2.2 mmoles) of triethylarnine was introduced. After 5 minutes. 0.3 ml (2.2 mmoles) of trimethylchlorosilane was added to the mixture and stirring was continued for 1 hour at room temperature. The mixture was cooled again and, after addition of another equivalent of triethylamine. 3-(2,6- dichlorophenyl)isoxazol-S-yl-acetyl chloride (prepared as described in Example I) in 5 ml of ethyl acetate was added drop-wise to the reaction mixture with the temperature being maintained below 5C.

After the addition. the ice-bath was removed and the reaction mixture was stirred for another two hours at room temperature. Then it was poured into a mixture of ml of water and 30 ml of diethyl ether with ice cooling while the pH was kept at 7.0. The aqueous layer was washed with another portion of diethyl ether (30 ml) and ethyl acetate (30 ml). After addition of 50 ml of ethyl acetate, the aqueous layer was acidified to pH [.7 and the layers were separated and the aqueous layer was extracted again with 50 ml of ethyl acetate. The combined ethyl acetate layers were washed once with acidified ice-water at pH L5 and twice with icewater. After separation, drying over magnesium sulfate and treatment with Norit, the ethyl acetate layer was concentrated to about one third of its volume and then sodium a-ethylcapronate was added. The precipitated sodium salt was washed once with ethyl acetate and twice with n-hexane and after filtration dried in vacuo to obtain 438 mg (0.8 mmoles 44%) of the sodium salt of 7-[ 3-( 2,6-dichlorophenyl)-isoxazol-5-yl 1- acetamido-cephalosporanic acid. According to TLC, the compound was pure.

A partial analysis of the lR-analysis of the final product (KBr disc. values in cm) 1760 C Ofi-lactam and C O ester l690 1670 C O amide i600 C O carboxylate ion I558 C =C orC= N 1230 COCester I025 782 C-Cl Analysis of the PMR spectrum of the final product dissolved in hexadeuterodimethylsulphoxide (6O Mejivalues in ppm, tetramethylsilane as an internal standard).

C 3 7.55 sharp narrow (3 protons) splitting pattern NH 9.22 and 9.35 doublet (J'==8 cps.

1 proton) Elementary analysis for C H N O1SCl Na V2 H O.

Found Average Calculated (with 2 mole of crystalwater) C 44.97 45. if) 9% 45.03 7: 45.26 K H 3.3l -38 3.34 3.08 7? N 7.70 7.72 it 7.71 X 7.54 72 S 5.70 5.67 1' 5.68 71 5.75 1' EXAMPLE [V Sodium salt of 7- l 3-( 2,6-dichlorophenyl )isoxazol-S- yl]acetamido}desacetoxycephalosporanic acid 0.46 ml (3.3 mmoles) of triethylamine were added to a suspension of 620 mg (2.9 mmoles) of 7- aminodesacetoxycephalosporanic acid in 10 ml of ethyl acetate in a 50 ml three-necked vessel. equipped with a gas inlet tube. a thermometer and a dropping funnel, under a nitrogen atmosphere and stirred mechanically. after cooling of the suspension with an ice-bath. After 5 minutes. 0.42 ml (3.3 mmoles) of trimethylchlorosilane was introduced and stirring was continued for one hour without external cooling. Then the reaction mix ture was cooled again with ice-bath and another 0.41 ml (2.9 mmoles) of triethylamine was added. 3-(2.6- dichlorophenyl) isoxazol-S-ylacetyl chloride (prepared as described in Example 1) in 5 ml of ethyl acetate was introduced drop-wise to the reaction mixture at such a EXAMPLE V 6-{ 3-( 2.6-dichlorophenyl)-4-carboxyisoxazol-5- yllacetamidolpenicillanic acid poon r H H a s l o. a a

o I |,H 3

c (7 0 coon In a three-necked flask equipped with a gas inlet tube, thermometer and dropping funnel, 314 mg 1.0 mmoles) of trimethylsilyl 6-isocyanatopenicillanate and 3 16 mg 1.0 mmoles) of 3-(2,6-dichlorophenyl)-4- carboxyisoxazol-S-yIacetic adid (prepared by the reaction of 3-(2.6-dich1orophenyl)-4-carboxy-5-methylisoxazole formed via a 1,3-dipolar addition of 2,6- dichlorophenyl benzonitriloxide and trimethylsilyl 2- butynoate with 2 equivalents of n-butyl lithium and one equivalent of tetramethylethylenediamine in toluene followed by carboxylation with CO were dissolved in 25 ml of benzonitrile. To this mixture, a solution of 145 25 rate that the temperature did not rise above C. The g O B 0f N-methylbenllmldalole III 5 m] of li b h was removed d i i was Continued benzonitrile was added dropwise and there was a direct f another two h formation of carbon dioxide. After two hours, the car- The reaction mixture was then poured into a mixture bon dioxide evolution ceased, and the reaction mixture of ml of water and 20 ml of diethyl ether with icewas Poured into a mixture of 30 ml of Water and cooling and mechanical stirring while the pH was main- 50 m1 of dlethyl ether with lcer'zoollng. the P being tained at 7.0. The aqueous layer was washed once wi h maintained at 7. The aqueous layer was extracted twice 20 ml of ethyl acetate and once with diethyl ether (20 e With 50 1111 Of ie hyl therml). After addition of 40 ml of ethyl acetate to the After addition of 50 ml of diethyl ether and 10 ml of aqueous layer, the pH was brought to 1.7. The aqueous ethyl acetate, the pH was brought to 4. The layers were layer was extracted once again with 30 ml of ethyl aceseparated and the aqueous layer was extracted twice tate and then these layers were combined and washed with 50 ml of diethyl ether. The combined organic layonce with 20 ml of acidified ice-water (pH 1.7) and ers were washed with ice-water and dried over magneonce with normal ice-water (20 ml). After drying over sium sulfate. After removal of the solvent, 136 mg of magnesium sulfate and treatment with Norit, the ora slighly yellow solid ws left behind, which was pure 6- ganic layer was concentrated to about one third of its [3-(2,6-dichlorophenyl)-4-carboxyisoxazol-5- original volume. Sodium a-ethylcapronate was added yl]acetamido penicillanic acid according to TLC. and the precipitated sodium salt was collected on a fi1- A partial analysis of the lR spectrum of the final ter, washed with ethyl acetate and with diethyl ether product (KBr disc, values in cm). and dried in vacuo to obtain 0.603 mg 1.23 mmoles 43%) of the sodium salt of 7-[3-(2,6-dichlorophenyl- 1775 C O B-lactam )lsoxazol-5yl l-acetamtdo desacetoxycephalosporamc 1700 C 0 carboxy] acid. According to TLC, the compound was pure. I6 C =C aromatic A partial analysis of the IR spectrum of the final {23 8 g z j ring product (KBr disc, values 111 cm). 780 C -C1 I 3400 NH (broad absorption) Analysis of the PMR spectrum of the final product dis- :23 C i 0 B-lagtam solved in hexadeuterodimethylsulphoxide (60 Me, 5 I590 g;8 $L: ion -values in ppm, tetramethylsilane as an internal stan- 1555 c=c dard). I 1540 NH def. (shoulder) CCH.t 1.52 and 1.65 (6 protons) Analysis of the PMR spectrum of the final product CllS- solved in hexadeuterodimethylsulphoxide Mc.8- cHzcand CF 432 (3 protons) values in ppm. tetramethylsilane as an internal stan- 60 0,4; and C..- H 533-5 multiple! (2 protons) dard) CH, 7.55 sharp narrow splitting pattern NH 9.10 doublet C -CH 1.98 3 protons) S-CH: 2.95 3.65 AB-quartet (3* 17.5 cps;

2 protons) EH, d C H 22.98 97 (2 p4rotons) 2 an .8R.4. (.1-= .5 cps; protons) and 5.43-5.52 EXAMPLE VI iwxazo1--C,H 6.50 (1 proton) Cu n 7.55 sharp narrow (3 protons) Sodium salt of 6-{[3 (2.4.6- trnnethyl) splitting pattern Phenylisoxazol-S-yllacetamidolpenicillanic acid.

c CH3 a COONa using the procedure of Example ll, L23 g mmol) few minutes at 0C followed by removal of the ice-bath.

Of 3-( 2.4.6-trimethyl )-phenyl-isoxazol-5-yl-acetic acid were reacted with 1.57 g (5 mmol) of trimethylsilyl 6 isocyanato-penicillanate in ml of dry dichloromethtrue in the presence of about 0.05 ml of N-vinylimidazole (catalyst). The reaction was finished after 6.5 hours and according to thinlayer chromatography. the isocyanate was converted in the desired product to the extent of about 60%. The reaction product was treated in the usual fashion. At pH 4.5, the penicillin was extracted from water with diethyl ether and the 50 lution in ether was washed with a small volume of icewater, treated with activated charcoal, dried over anhydrous magnesium sulfate and concentrated in vacuo to some extent at 0C.

A solution of sodium a-ethylcapronate in diethyl ether was added drop-wise to the concentrated solution and the colorless precipitate formed was recovered by filtration and washed repeatedly with cold diethyl ether. After drying in vacuo. the product, sodium salt of 6- 3-( 2,4,6-trimethyl )phenylisoxazol-S- yllacetamido}penicillanic acid. weighed 0.8 g. Good purity of the product was indicated by thin-layer chromatography. IR and PMR spectra.

Analysis of the PMR spectrum of the final product dissolved in a about 2:l mixture of hexadeuterodimethylsulphoxide and D 0 (60 Mc,5-values in ppm, internal reference 2,Z-dimethylsilapentane-S-sulphonate):

6.95 (singlet. 2 protons) 6.3 (l proton) about 5.45 lAB-quartet.5 0 l ppm.

J 4 cps. 2 protons) 2.8 ml (20 mmol) of triethylamine were added dropwise to a stirred suspension of 2.7 g (10 mmol) of lamino-cephalosporanic acid in ml of dry dichloromethane at 0C. Next, 2.55 ml (20 mmol) of trimethylchlorosilane were added drop-wise and after completion of the addition. the reaction mixture was held a l l 0 b n c p a Stirring was continued for 1 hour at room temperature. Subsequently, 1.2 ml (10 mmol) of quinoline were added, followed by the drop-wise introduction of a solution of approximately l0 mmol of 3-(2,4.6- trimethyl)phenylisoxazol-5-yl-acetyl chloride in 20 ml of dry dichloromethane at 5C. After a few minutes additional stirring at room temperature, the reaction mixture was poured into ice water followed by addition of dilute sodium hydroxide. At pH 7 the layers were separated and the water-layer twice was extracted with diethyl ether. The organic layers were discarded and the water-layer between pH 5 and pH 1 was repeatedly extracted with diethyl ether. The organic layers were separately in spected by thin-layer chromatography. The cleanest extracts were combined, washed with ice water. dried over anhydrous magnesium sulfate, filtered, concentrated somewhat in vacuo and finally treated with a solution of sodium a-ethylcapronate in ether. The solid precipitate was collected by filtration, washed with diethyl ether and dried in vacuo to constant weight to obtain 2.5 g of the sodium salt of 7-H- (2,4,6-trimethyl )phenylisoxazol-S -yl l-acetamido-cephalosporanic acid. in order to obtain the crystalline monohydrate, the crude product which according to thin-layer chromatography did not contain other sulphur containing substances was crystallized from acetone. The final product (I g) was pure except for the presence of a small amount of acetone, and it contained one mole of water per mole of water per mole of cephalosporin.

Analysis of the PMR spectrum of the final product dissolved in hexadeutero-dimethylsulphoxide Mc B-values in ppm, internal reference 2.2-dimethylsilapentane-S-sulphonate):

N-H 9.26 and 9,12 (doublet, J=8.5 cps.

about 0.8 proton) C ll: 6.93 (slightly broadened singlet.

2 protons) 6.33 (singlet. l proton) 5.66. 5.58. 5.52 and 5.44 (slightly broadened signals. .I=8.5 cps and (iOONa .I ,,,=4.7 cps. l proton) 5.04 and 4.96 U =4.7 cps) C.t-H 13 prots.

OCH- 5.20. 4.99, 4.92 and 4.7l(.l

l2.5 cps) CH CO 3.92 (broadened singlet. 2 protons) S-CH -3.72.-3.43.-3.33 and-3.04

(broadened signals. AB-quartet.

l =l7.5 cps. 2 protons) 3500 OH carboxyl pCH 2.27 (3 protons) I 3300 NH (oCH;,)- 2.07 (singlet) I772 B-lactam protons I730 C=O carhoxyl O-COCH;L 2.01 (singlet) I690 C==O amide 5 I380 143i) isoxazole ring absorptions EXAMPLE VIII EXAMPLE Cyclohexylamine salt of 7{[3-(2.6(4-methyl-isoxazol-5-yllacetamido 6- {a-chloro-l3-(2,4,6-trimethyl) phenyl-isoxazol-S-yl] desacetoxycephalosporaniclacid. l0 acetamidolpenicillanic acid. Cl

/ l i' R H it c. fa

m 0:5 11 j on f coon ca. H o s j E I L L f "t: CH

\CHB (U, 81 s i I 3 2 A solution of approximately l mmol of trimethylsilyl A solution of 700 mg of trimethylsilyl o-isocyanato- 7-isocyanato-desacetoxycephalosporanate in 2 ml of penicillanate in l0 ml of dry dichloromethane was toluene was added to 286 mg (1 mmol) of 3-(2.6- added drop-wise over 25minutes to a solution of 700 dichloro)phenyl-4-methyl-isoxazol-5-yl-acetic acid mg of l-chloro-1-[3-(2,4,6-trimethyl)Phenyl-isoxazolpartly dissolved in l0 ml of dry toluene. Introduction 5-yl] acetic acid and about 0.03 ml of N-vinylof approximately 0.1 mmol of l-isopropylimidazole (catalyst) In 25 ml of dry dichloromethane. benzimidazole (catalyst) brought about the onset of a The reaction mixture was additionally stirred during 4 slow reaction (duration about 24 hours at room temhours. ln situ hydrolysis of the silylester was achieved perature). When the liberation of carbon dioxide was by the addition of about 0.2 ml of ethanol at 0C. The no longer noticeable in the stream of dry nitrogen reaction mixture was poured intoawell stirred mixture passed over the surface of the stirred reaction mixture. of diethyl ether and iced water buffered to pH 7. After the contents of the vessel were poured into a well separation of the layers, the water-layer was once more stirred mixture of iced water and diethyl ether. The pH extracted with diethyl ether and subsequently acidified was brught to 6.8 and the layers were separated and the to a pH Of 3.5. The desired compound was incomwater-layer was twice extracted with diethyl ether. pletely removed from the water layer by two extrac- The combined organic layers were twice washed with tions with diethyl ether. These extracts were combined, iced water. The organic layer was discarded and the washed with a small volume of iced water. dried over combined water-layers (70 ml) were extracted at pH anhydrous magnesium sulfate and evaporated in vacuo. 2.3 with 80 ml of a 2:l mixture of diethyl ether and The residual oil was dissolved in 5 ml of acetone and ethylacetate. This extract was twice washed with 5 ml a dilute solution of cyclohexylamine in diethyl ether of iced water. dried over anhydrous magnesium sulfate, was added slowly until no further increase of precipifiltered and completely evaporated in vacuo. The residtate was noticed. The coloreless solid was collected by ual, slightly yellow oil solidified when stirred with dry filtration. washed with cold diethylether and dried in diethyl ether. The ether was decanted and the solid vacuo to obtain 250 mg of the cyclohexylamine salt of again stirred twice with ether. The almost colorless 6 hlom-[3-(2,4,6-trimethyl)phenylisoxazol-S-yl] Solid Was dried in VaCUO t0 Constant weight to Obtain acetamidol-penicillanic acid. The identity of the final 290 g of l )p y y product was confirmed by PMR spectra and lR spectra isoxazol-5-yl1acetamidol-desacetoxycephalosporanic (KB;- di 1775 C=O id 1390 d 1450 acid. The final product was examined by thin-layer cm": isoxazole ring). The purity of the final product chromatography which indicated the presence of only was estimated to b about 35%,

one sulphur containing compound. The alleged structure was confirmed by IR and PMR spectra. The PMR spectrum revealed that the final product was about 82% pure since it consisted of 1 mole of the starting EXAMPLE X acetic acid and 2.5 moles of diethyl ether (probably crystal bound) on 5 moles of the desired product. Sodium salt of Partial analysis of the IR spectrum of the final prod- 6-{[3-(2,6-dichloro)phenyl-4-methyl-isoxazol-5-yl] uct (in chloroform, values in cm): acetamidolpenicillanic acid.

1 7 18 C1 LIT-1K h S C C r H n c cn N. CH. CH (CH COONa Using the procedure of Example ll. 286 mg l mmol) Ct tCl-tm I54 and 1.64 (6 protons) of 3-( 2.6-dichloro)Phenyl-4-methyl-isoxazol-S-yl- 1CH;: .ggoztgrltzgldened Single) acetic acid were reacted with 314 mg (1 mmol} of tn- C H 4m (3 protons) methylsi lyl 6-lsocyanato-pentclllanate in ID ml of dry ZI and C'; H Bo-u 55 (multiplet 2 protons) toluene 1n the presence of about 0.0l ml of N-isoprois 1,44 about 7,6 (sharp n rr w splitting pyl-benzimidazole (catalyst). P 3 Prolons) The reaction of the mixture was stirred over night at lbom (douhlet 09 Fromm) about C and according to a thin-layer of chromatogram. the isocyanate could have been converted into the desired product for at least 75%. The reaction EXAMPLE XI product was treated in the usual manner. At pH 3.8. the Cyclohexylamine salt of penicillin was removed from the water-layer by three 6{a-methyl-[3-(2.4.6-trimethyl)Phenyl-isoxazol-S- 40 ml extractions with diethyl ether. The combined exyllacetamidolpenicillanic acid.

\ C CH O H coon. HQN Q tracts were washed with ice water, dried over anhy Using the procedure of Example ]X, a reaction with drous magnesium sulfate, filtered and concentrated in equimolar amounts of trimethylsilyl 6-isocyanato pe i vacuo to a volume of about 5 ml. Addition of a solution cillanate and l-methyl-l [3-(2.4,6-trimethyl)phenyl- CH O=C N 0f a-ethyleapl'ohate in diethyl ether resulted in isoxazol-Syllacetic acid was carried out in the presence a colorless precipitate which was recovered by filt 'aof a small amount of N-vinyl-imidazole using as solvent, Ii0n, was Washed With Cold diethyl ether and dried in dry dichloromethane. The conversion was completed Vaello t0 Constant Weight Finally, the F Was after 6 hours stirring at room temperature and the reac- Urated in a Small Volume of cold y acetone to obtain 40 tion mixture was treated in the usual fashion. In the iso- 300 g the Sodium salt of 'i[ y lation procedure, the pencillin was extracted at pH 4 4-methyl-lsoxalol-5 yllaeelamido -Pehiemahie acidwith diethyl ether and finally obtained as its cyclohexyl- Examination of the final Product by min-layer Chroma amine salt. Thin-layer ehromatograms. the IR spectrum tegraphy. IR Spectra and PMR Spectra confirmed the (intensive B-lactamcarbonyl absorption at I778 cm structure. The product was contairninated with only disc) and the p spectrum fi d h struc- Small amounts of acetone and sodium aethylca ture of the final product and indicated its good state of P purity. Partial analysis of the complicated PMR spec- Analysls 0f the PMR Spectrum of the final Pmduet trum of the final product (a mixture of the D- and the dissolve m heXadeutefodlmelhylsulphoxlde 9 0 L-isomer) dissolved in hexadeuterodimethylsulphoxide values in ppm, internal reference 2.2ldlmethylsrlapent- 5 (6O 5 in ppm intern-1| reference 22- ane-5-Sulph0nat dimethyl-silapentane-S-sulphonate):

N-H 8.9 (about 1 proton) C H. 6.95 (somewhat broadened singlet.

2 protons) isoxazolyl C H 6.35 (2 close singlets. I proton) -H and C,.H about 5.4 (2 protons) C,.-H about 4.2 (diffuse quartet) 2 protons .H about 4.0 (2 close singlets) cyclohexyl -H about 2.9 (broad absorption area.

about I proton) pCH; 2.3 (singlet. about 3 protons) roughly about (oCH; l- 2.1 (singlet. about 6 protons) 26'prot.

cyclohexyl C,-.H about 0.9 -+about 2.3

EXAMPLE X11 Sodium salt of 7{ 3-( 2.4,6-trimethyl )phenyl-4-methyl-isoxazol-5- yl lacetamido}cephalosporanic acid.

Analysis of the PMR spectrum of the final product dissolved in an about 2:1 mixture of hexadeuterodimethylsulphoxide and D (60 Mc.6-values in ppm. internal reference 2.Z-dimethylsilapentane-S-sulphonate):

(1.95 lsinglet. 2 protons) 5.72 and 5.64 (doublet. J=-L6 cps.

l proton) 5.10 and 5.02 (doublet. 1 41) cps) about 5.15. 4.92. 4.81 and 4.59

(AB-quartet. J'- 13.2cps) about 3.55 (center of AB quartet) 3.87 (somewhat broadened singlet) 2.28 (3 protons) 2.05 (singlet) 3 protons 4 protons protons 1.98 (single!) 17] (3 protons) ZOONa l .38 ml 10 mmol) of triethylamine were added drop-wise to a stirred suspension of 1.3 g (5 mmol) of 7-amino-cephalosporanic acid in ml of dry dichloromethane at 0C. Next. 1.26 ml 10 mmol) of trimethylchlorosilane were added drop-wise at 0C and after completion of the addition of trimethylchlorosilane. the reaction mixture was stirred for a few minutes at 0C followed by removal of the ice-bath. Stirring was continued for 1 hour at room temperature and then 0.6 ml (5 mmol) of quinoline were added followed by the drop-wise introduction of a solution of approximately 4.5 mmol of 3-(2.4,6-trimethyl)phenyl-4-methyl-isoxazol-5-yl-acetyl chloride (in about 90% purity prepared from 1.3 g (5 mmol) of the corresponding carboxylic acid) in 10 ml of dry dichloromethane at 5C. After a few minutes additional stirring at room temperature, the reaction mixture was poured in ice water. The pH was raised to 7 and the layers were separated. The water-layer, containing according to thin-layer chromatograms one main reaction product (a small amount of 7-amino-cephalosporanic acid) and a small amount of a by product (possibly the ri -isomer of the desired product), was washed twice with diethyl ether. The organic layers were discarded and the water-layer was successively extracted at pH 5.0. 4.5 and 4.0 with diethyl ether. The extract of pH 4.0 contained only the desired main product. Addition of a solution of sodium a-ethyl-capronate to this extract gave a colorless solid precipitate for 1.2 g of the sodium salt of 7-{[3-(2.4,6- trimethyl)phenyl-4-methyl-isoxazol-S-yl]acetamido cephalosporanic acid. According to thin-layer chromatograms. IR and PMR spectra the final product was only contaminated by small residual amounts of diethyl ether (about 1% by weight).

EXAMPLE Xlll Sodium salt of v 7- l 3-( 2,4,6-trimethyl )phenyl-4-methyl-isoxazol-5- yl]acetamidoldesacetoxycaphalosporanic acid.

coona Using the procedure of Example 1X, a reaction was carried out between 1.3 g (5 mmol) Of 3-(2,4,6- trimethyl) phenyl-4-rnethyl-isoxazol-S-yl-acetic acid dissolved in 25 ml of dry dichloromethane and 5.04 pl mmol of trimethylsilyl 7-iso-cyanato-desacetoxycephalosporanate dissolved in 9 ml of toluene in the presence of about 0.05 ml of N-vinyl-imidazole (catalyst). The addition of the solution of the isocyanate in toluene took about 20 min. Evolution of carbon dioxide was already noticeable after 5 min. and after 7.5 hours additional stirring the reaction was interrupted since a thin-layer chromatogram of the reaction mixture indicated conversion of the isocyanate for about in the desired direction and evolution of carbon dioxide had almost stopped. The reaction mixture was treated in the usual fashion. The cephalosporin was extracted at pH 4.5 with a 9:1 mixture of diethyl ether and ethyl acetate and the combined extracts were washed with iced water, dried over anhydrous magnesium sulfate, filtered and completely evaporated in vacuo. The residual oil was dissolved in diethyl ether. Addition of a part of the estimated necessary amount of sodium a-ethylcapronate dissolved in diethyl ether resulted in a precipitate which was recovered by filtration and was washed with a small amount of cold diethyl ether, and dried in vacuo. Sodium a-ethylcapronate was again added to the combined filtrates. The resulting second crop of solid material was treated like the first crop. The third and last crop was obtained by adding dissolved sodium a-ethylcapronate till no further increase of precipitate occurred. The third crop being practically pure according to thin-layer chromatography was crystallized from acetone. Finally. the three crops were dissolved together in acetone. The acetone solution was somewhat concentrated in vacuo and subsequently seeded. After crystallization had subsided. the flask was placed in the refrigerator. The next day the crystals were recovered by filtration, were washed with cold acetone and diethyl ether and dried in vacuo to constant weight to obtain 1.7 g of the sodium salt of 7 [3-(2.4.6-trimethyl)phenyl-4 -methyl-isoxazoI-5- yllacetamido desacetoxycephalosporanic acid. The structure was confirmed by IR and PMR spectra. According to PMR spectra and thin-layer chromatograms. the final product was contaminated only by a very small amount of acetone and a small amount of N,N'-didesacetoxycephalosporanic acid urea.

Analysis of the PMR spectrum of the final product dissolved in an about 2:1 mixture of hexadeuterodimethylsulphoxide and D 0 (60 Mc,6-values in ppm, internal reference 2.2-dimethyl-silapentane-5-sulphonate):

CH: 6.97 (slightly broadened singlet.

2 protons) 5.63. 5.46. 4.97 and 4.90 (ABquartet. J= 4.5 cps. 2 protons) -H and Ctr-H (H -CO 3.86 (broadened singlet. 2 protons) SCH about 3.7' 2.9 (AB-quartet. J=

17.5:1 cps. 2 protons) p-CH; 2.29 (3 protons] [o-CH)2 L98 (singlet) 9 protons -Clix 1.94 (singlet) 3 isoxazol-C,CH;, |.7l (3 protons) EXAMPLE XIV 6-1[3-(2.6-dichloro)phenyl-4-carbamyl-isoxazol-S- yllacetamido} penicillanie acid.

ant:

layer chromatogram of the reaction mixture indicated 65 good conversion of the isocyanate. The reaction mixture was poured into a well stirred ice-cold mixture of 30 ml of water. ml diethyl ether and 20 ml of ethyl acetate. Dilute sodium hydroxide was added till pH 8.5.

The layers were separated and the water-layer was purified by extraction with diethyl ether. The organic layers were discarded and the water-layer at pH 3.0 was extracted with a lzl mixture of diethyl ether and ethyl acetate. The combined extracts were washed with icedwater, dried over anhydrous magnesium sulfate, filtered and completely evaporated in vacuo. The resulting. slightly yellowish solid 1.1 g) was examined by IR and PMR. The product contained the desired 6-{[3- (2,6-dichloro )phenyl-4-carbamyl-isoxazol-S- yl]acetamido penicillanic acid and also minor amounts of N,N'-di-penicillanic acid urea and the starting carboxylic acid. In order to obtain a more pure sample. the crude product was repeatedly extracted with cold-dry diethyl ether in which the urea is slightly soluble. The ethereal extract was mixed with iced water buffered to pH 7 and the greater part of the starting carboxylic acid was removed from the water-layer at pH 4.5. Finally. the water-layer was repeatedly extracted at between pH 4.5 and pH 3.5 with mixtures of much diethyl ether and small but gradually increased amounts of ethyl acetate. Extracts free from the starting carboxylic acid. the urea and degradation products were combined and after the usual manipulations were completely evaporated in vacuo. The resulting colorless solid was dried to constant weight to obtain 350 mg of product. According to thin-layer chromatograms, IR and PMR spectra the final product was pure except for the presence of slight residual amounts of ethyl acetate and diethyl ether. The IR spectrum (KBr disc). complicated by the monomer-dimen feature, i.a. showed a broad intensive area between 3000 and 3600 cm with peaks at 3450, 3350 and 3200 cm ascribable to NH absorptions of both amide groups, a carboxyl OH absorption at about cm". a broad very intensive coon carbonyl absorption area with peaks at i 1790, i 1725. :t [695 and i I655 cm ascribable respectively to the B-lactam, the carboxyl, the CO.NH and the CO.NH group.

EXAMPLE XV Sodium salt of 6-{l3-(2,6-dichloro)phenyl-4-cyano-isoxazol-5- yllacetamido l penicillanic acid.

297 mg (l mmol) of 3-(2.6-dichloro)phenyl-4- cyano-isoxazol-S-yl-acetic acid, 314 mg (l mmol) of trimethylsilyl 6-isocyanato-penicillanate and a trace of N-isopropylbenzimidazole were dissolved in 5 ml of dry dichloromethane. According to a thin-layer chromatogram. good conversion of the isocyanate was reached after 3 hours reaction at room temperature and then the reaction product was treated in the usual fashion. In the isolation procedure. the solution of the penicillin in water was purified by extractions with diethyl ether at pH 7.0 and 4.5. The penicillin was removed from water by extraction with diethyl ether at pH 3.3 and the ethereal extract was washed with iced water. dried over anhydrous magnesium sulfate. filtered and evaporated in vacuo. The residual oil was dissolved in about 3 ml of dry ethyl acetate followed by the addition of about 0.6 mmol of sodium a-ethylcapronate dissolved in a small volume of ethyl acetate. Addition of dry diethyl ether resulted in a slightly colored precipitate which was recovered by filtration, was washed with cold diethyl ether and dried in vacuo to constant weight to obtain l8O mg. The final product was examined as usual and it contained the sodium salt of 6-{[3-(2.6- dichloro)phenyl-4-cyano-isoxazol-S-yl]acetamido penicillanic acid and slight amounts of a degradation product and of sodium a'ethylcapronate. The IR spectrum of the final product (KBr disc) exhibited i.a. absorptions at 2280 (C a N), 1778 (carbonyl B-lactam). I690 (carbonyl amide), I610 (carbonyl carboxylate ion) and I 1400 cm (isoxazole ring absorptions).

EXAMPLE XVI Sodium salt of 6-{[3-( l )adamantyl-isoxazol-S-yl]acetamido penicillanic acid.

I c ca Using the procedure of Example 1x, a solution of 780 40 mg (2.5 mmol) of trimethylsilyl o-isocyanatopenicillanate in 10 ml of dry dichloromethane was added drop-wise to a solution of 650 mg (2.5 mmol) of 3( l )-adamantyl-isoxazol-5-yl-acetic acid and about 0.02 m] of N-vinyl-imidazole in ml of dry dichloromethane. The conversion was finished after a total of 2.5 hours stirring at room temperature as indicated by a drastic diminishment of evolution of carbon dioxide. A thin-layer chromatogram indicated good conversion of the isocyanate into the desired penicillin and the reaction product was treated as usual. in the isolation procedure, the penicillin was extracted from water by two extractions with diethyl ether. one performed at pH 5.5 and the other at pH 4.0. The extracts were separatedly washed with iced water. dried over anhydrous magnesium sulfate. filtered and completely evaporated in vacuo to obtain yields of 700 and 300 mg. respectively. Both products gave satisfactory IR spectra and contained according to thin-layer chromatography only one penicillin. Since the sample obtained by extraction at pH 5.5 was contaminated by the starting acetic acid derivative, it was dissolved in ether followed by addition of sodium a-ethylcapronate. The obtained sodium salt (350 mg) of 6-{[3-( l )adamantyl-isoxazol-S- yl]acetamido} penicillanic acid was pure except for a slight amount of residual sodium a-ethylcapronate. According to a PMR spectrum the second product was pure except for a slight amount of diethyl ether (about 4.0% by weight).

Partial analysis of the IR spectrum of the sodium salt of the final product (KBr disc, values in cm):

1: 3400 NH I605 C=O carboxylate ion 2910 :1520 NH def.

2853 C H groups 1405 isoxazole ring absorption 1775 C=O lllactam I675 C=O amide Analysis of the PMR spectrum of the final product (the acid) dissolved in hexadeuterodimethylsulphoxide (60 Mc,5-values in ppm, internal reference 2.2- dimethyl-silapentane-S-sulphonate):

c(ca

l H 3 3 c/ COONa about 8.9 (about 0.8 proton) 6.26 l proton) SSS-+5.60 (multiplet. 2 protons) -H 4.26 (l proton) 0zCH 3.79 (broadened singlet. 2 rotons) h] adamantyl group 1.90 and L73 (centers of some ga what broadened absorptions) a m protons C:|(CH:|]: L54 and l.5l

EXAMPLE XVll 6-{ a-p-nitro-benzyloxycarbonylamino-[ 3-( 2,6- diehloro)phenyl-isoxazol-Syl] acetamidolpenicillanic acid.

it H

\ f NH CH C Mgr-r mt owe. L OOH 2.33 g mmol) nitro)benzyloxycarbonylaminol 3-( 2,6- dichloro)phenyl-isoxazol-S-yllacetic acid. 1.57 g (5 mmol) of trimethylsilyl 6-isocyanato-penicillanate and 0.1 ml of N-vinyl-imidazole (catalyst) were dissolved in 5 50 ml of dry dichloromethane. After 3 hours stirring under nitrogen at room temperature the conversion was completed and according to thin-layer chromatography. the isocyanate could have been converted for about 70% into the desired product. The reaction product was cooled down to 0C followed by the addition of a few ml of cold acetone containing enough water to hydrolyze the silylester. Next. the mixture was completely evaporated in vacuo in the cold and the residue was dissolved in 75 ml of a cold 1:] mixture of diethyl ether and ethyl acetate. Since it was intended to use this penicillin for the preparation of the penicillin of Example XVIII, the isolation procedure was not aimed at the isolation of the product in a substantially pure state but instead directed at the isolation of as much as possible of the desired product. Therefore, the solution was mixed with 70 ml of iced water buffered to pH 7. The well stirred mixture was acidified to pH 5.8 and transferred to a separatory funnel. The water-layer was removed and discarded since it contained the by product N.N'-di-penicillanic acid urea and merely traces of the desired product. The organic layer was then washed twice with slightly acidic ice-cold water and once with a small amount of neutral water. The organic layer, in this way completely freed from the urea and the catalyst, was dried over anhydrous magnesium sulfate, filtered and completely evaporated in the cold.

The residue was dried in vacuo to constant weight to obtain 3.4 g of a slightly yellow, predominantly crystalline solid 6-{a-p-nitro-benzyloxycaronbylamino-[3- (2.6-dichloro )phenyl-isoxazol-S-yl]-acetamido} penicillanic acid. Thin-layer chromatograms of the final crude product indicated the presence of only the desired penicillin and the starting protected amino acid in about 2:1 ratio. This was confirmed by the PMR spectrum which also revealed the presence of ethyl acetate and a slight amount of water. The calculated amount of the desired penicillin in the crude product was 2.2 to 2.4 g.

of l p- EXAMPLE XVlIl 6- {a-amino-[ 3-( 2,6-dichloro)phenyl-isoxazol-S- yllacetamido} penicillanic acid.

3.0 g of the crude product of Example XVll containing about 2 g of 6-(a-(pnitro )benzyloxycarbonylaminol 3-( 2.6- dichloro)phenyl-isoxazol-S-yl]acetamido}penicillanic acid dissolved in ml of ethylacetate were mixed with 25 ml of water. The pH of the mixture was brought to 7.0 by addition of dilute sodium hydroxide and after the introduction of l.5 g of Pd/C 10%. hydrogen was continuously passed in beneath the surface. Thin-layer chromatography showed that the reduction was complete after min stirring at room temperature. For

, l0 minutes. nitrogen was passed through the reaction mixture. ice water added and the pH brought to pH 4.7. The contents of the funnel were transferred to a separatory funnel and the mixture settled to a clear ethyl acetate layer and a water layer separated by an emulsion layer. The water layer was removed and kept aside. Then, the emulsion layer was centrifuged and the resulting layers were separated. The ethyl acetate layer was combined with the first ethyl acetate extract and the water layers were also combined and extracted once with ethyl acetate. The water layer was discarded and remaining catalyst was removed from the collected ethyl acetate extracts by filtration. The colored filtrate was concentrated in vacuo at 0C to a volume of about 25 ml and I00 ml of iced water were added and the mixture was brought to pH 7.0. The layers were separated and the colored organic layer was discarded. The solution of the desired compound in water was purified by two extractions with a 1:1 mixture of ethyl acetate and diethyl ether. The resulting, practically colorless solution in water was acidified to pH 4.7 and was extracted twice with an excess volume of ethyl acetate. The water layer was discarded and the combined ethyl acetate layers were washed twice with a small amount of iced water. Thin-layer chromatograms of the final extract showed one elongated (the compound is a D.L- mixture) sulphur and ninhydrin positive spot. After complete evaporation of the extract a slightly colored solid of 650 mg of dry material was obtained. The final product was examined by IR and PMR spectra and was found to a l:l molar mixture of the desired 6-(01- amino-[ 3-( 2,6-dichloro)phenyl-isoxazol-S- yllacetamido}penicillanic acid and ethyl acetate possibly contaminated by slight amounts of a 3-(2,6- dichloro)phenyl-isoxazole-derivative.

Partial analysis of the lR spectrum of the final product (KBr disc, values in cm):

about 3300 NH about 2600 OH carboxyl l 780 (=0 B-lactam 1730 (=0 ethylacetate r. 1705 C=O carboxyl 1690 C=O amide and 1440 isoxazole ring absorptions EXAMPLE XIX Sodium salt of 6-{ 3-p-nitrophenyl-isoxazol-5-yl]acetamido penicillanic acid.

27 28 O N CH O H t1\O/c "c11 e lt ca c (gH OONa in the usual manner. a reaction was effected with 166 C-H 795- 24.4 (AA'BB' splitting pattern. 4 protons) mg (0.67 mmol) of 3p-nitrophenyl-isoxazol-5ylfig (L94 H prom") ZICCtiC acid, 210 mg (0.67 mmol) Of trimethylsilyl-o- (5-H and C.H 5.5 (slightly broadened singlet. 2 protons) C-H 4.l5 l proton) isocyanatopenicillanate and a trace of N-isopropylbenzimidazole, with a solvent of 5 ml of benzonitrile. The reaction was complete after 5 hours stirring at room temperature and the contents of the flask were poured into a ice-cold well stirred mixture of 25 ml of water, ml of diethyl ether and ml of ethyl actate. The acid mixture (pH3) was neutralized to pH 7 by addition of dilute NaOH. and the layers were separated. The organic layer was discarded and the water layer for purification was extracted once with ml of a l:] mixture of ether and ethyl acetate. 30 ml of a 1:1 mixture of ether and ethyl acetate were mixed with the water layer and the pH was lowered to 3.5. The layers were separated and the water layer was again extracted with 50 ml of the same solvent mixture. The combined organic layers were washed twice with a small volume of iced water. then dried over anhydrous magnesium sulfate, filtered and completely evaporated in vacuo. The resulting yellowish oil was triturated with dry diethyl ether. The resulting partly crystalline solid was recovered by filtration and then repeatedly stirred up in ether. After drying in vacuo, the final, colorless product weighed 73 mg. Inspection of the product by thin-layer chromatography and by PMR spectra indicated that the desired 6-{[3p-nitrophenyl-isoxazol- 5-yl] acetamido penicillanic acid contained 5 to 6 moles of water per mole of compound and a small amount of diethyl ether, but that it was virtually pure in other respects. The collected ethereal filtrate and washings were completely evaporated and the residue was dissolved in 3 ml ofa dry l:l mixture of ether and ethyl acetate and then treated in the cold with a dilute solution of sodium a-ethyl-capronate in ether. The precipitated sodium salt of the said penicillin was recovered by filtration and was repeatedly washed with dry ether. After drying, this product weighed 134 mg. The product was examined in the usual manner. Not counting adhering water (much less than in the case of the free penicillanic acid) the purity of the sodium salt was estimated to be about 80-85% since it contained about 5% by weight of a degradation product and l0- l5% by weight of sodium a-ethylcapronate.

Analysis of the PMR spectrum of 6-{ [3-pnitrophenyl-isoxazol-S-yllacetamido penicillanic acid dissolved in a mixture of about 6 parts of hexadeuterodimethylsulphoxide and 1 part of D 0 (60 Mc,6-values in ppm, internal reference 2.2- dimethylsilapentane-Ssulphonate)2 4,0 (somewhat broadened s' l t. 2 t CH (CH )2 ing e Pro 0115) 1.63 and [.52 (6 protons) Partial analysis of the IR of the sodium salt of 6-{[ 3- (4-nitro)phenyl-isoxazol-S-yllacetamido}penicillanic acid (KBr disc, values in cm):

1355 N0 (intensive) 858 C-NO and aromatic substitution pattern (medium intensities) EXAMPLE XX 6- {a-carbamyH 3-( 2.6-dichloro )phenyl-isoxazol-S- yl]acetamido }penicillanic acid.

A solution of 3.7 mmol of n-butyllithium in hexane was added drop-wise to a solution of 1.0 g (3.7 mmol) of 3-(2,6-dichloro)phenyl-isoxazol-S-yl-acetamide in 15 ml of dry tetrahydrofuran cooled down to C. The rate of addition was adjusted to reaction temperatures below 60C. After a few minutes additional stirring at 70C. 0.47 ml (approximately 3.7 mmol) of freshly distilled trimethylchlorosilane were introduced drop-wise. Afterwards the cooling bath was removed and the temperature was allowed to rise to 30C. This procedure addition successively of 1 equivalent of n-butyllithium and 1 equivalent of trimethylchlorosilane was repeated in the same fashion. To the now in situ prepared solution of N,N-bis-trimethylsilyl derivative of the starting product in a mixture of 15 ml of tetrahydrofuran and about 3.5 ml of Hexane 0.56 m] 3.7 mmol) of N,N,N'.N'-tetramethylethylene-diamine was added. The mixture was again cooled down to C and a solution of about 3.7 mmol of nbutyllithium in l.76 ml of hexane was added drop-wise. The rate of addition was adjusted to reaction temperatures of maximum 70C. The reaction mixture was additionally stirred for one hour at 70 to 60C. The sequence of reaction was completed by the dropwise addition of a solution of L16 g (3.7 mmol) of trimethylsilyl 6-isocyanato-penicillanate in ID ml of dry toluene whereby the reaction temperature was not allowed to rise above 55C. The reaction mixture was then COOH stirred at C for min. and the reaction mixture and dilute hydrochloric acid were slowly and simultaneously poured into a well stirred and icy-cold mixture of 50 ml of diethyl ether and 50 ml of water of pH 4. Then the pH ofthe mixture was raised to 7 and the layers were separated. The water layer was again extracted with 50 ml of ether at pH 7 and the organic layers were discarded. The water layer was extracted three times with ether successively at pH 5.0. 4.5 and 4.3 and once extracted with a 1:1 mixture of ethyl acetate and diethyl ether at pH 4.3 Thin-layer chromatography showed that the water layer no longer contained the desired penicillin accompanied with small amounts of sulphur containing impurities and that the first three ethereal extracts contained 6-{a-carbamyl-[3-(2,6- dichloro)phenyl-isoazol-S-yl]acetamidolpenicillanic acid in a substantially pure state. The ethereal extracts were combined, washed with iced water. dried over anhydrous magnesium sulfate. filtered and completely evaporated in vacuo. The obtained solid weighed 500 mg after prolonged drying in vacuo. [R and PMR spectraof the final product confirmed the alleged structure of the penicillin. The estimated purity was 80 -85%.

isoxazolyl C H 6.22

C,-,H and -H 5.50 (2 protons) -H 4.32 CH- .CO 3,82 (2 protons) isoxazolyl-CH; 2.23 :t :ll: L65 and L52 Partial analysis of the IR-spectrum of the final product (in KBr, values in cm):

23500 OH carboxyl I780 C=O B-lactam I740 (=0 carboxyl I670 C=O amide 1380-1430 isoxazole ring absorptions EXAMPLE XX" 7-{[ 3-methyl-isoxazol-5-yl lacetamido }cephalosporanic acid.

Using the procedure of Example [[1 3-methyI-isoxazol- Partial analysis of the IR spectrum of the final prod- 5-yl ac ylchl ri (pr p r m -5 mm f uct (KBr disc, values in cm): methyl-isoxazol S-yl acetic acid and thionyl chloride) 13440 NH (presumably of CCFNHZ) ylvas hrought into reaction with N,0-bis-trlmethyls|lyl 13330 NH (presumably ofCO-NH) -amm0 cephalosporanate (prepared from 1.224 mg :3210 NH (presumably bonded NH) (4.5 mmoles) of 7-ACA. After the reaction and work- 2338-2650 ing up of the reaction mixture 790 mg (44 of a 11720 (=0 (carboxyl) slightly yellow coloured product, 7-{[3-methyli228 C=O of CO NH and Co NH2 isoxazol-5 -yl]acetamido cephalospor anic acid was 1598 C=C aromatic and NH2 deformation isolated Wlth 8. purity about 70 according IO TLC. IR- :[525 presumably NHbdeforrnation d pMR i33 I430 Flflij'fifighifii lflli n Analysis of the PMR-spectra of the final product dispattern. solved in a mixture of deuterochloroform and hexadeutero DMSO with some D 0 added (60 Mc, fi-val- EXAMPLE XXI ues in ppm, tetramethylsilane as an internal standard):

6-( [3-methyl-isoxaz0l-5-yllacetamidolpenicillanic isoxazolyl CFH M2 acid. CTH 5.75 and 566 (J 4.5 cps.

-H it i: ca c c 5 2" g CH 0 l 1. lat

proton) Using the procedure of Example ll 282 mg (2 mmol) CF" 505 and 497 U k 4,5 cps of 3-methyllsoxazol-5-yl acetic acid were reacted with l proton) 628 mg (2 mmol) of trimethylsilyl 6-isocyanatopenicillanate in 10 ml of dry dichloromethane in the CH2 Sig 5' U z 2 presence of three drops of N-isopropyl benzimidazole l 3.80-3.l5 (.I 18 cps, 2 (a catalyst). After the usual working up of the reaction g a mixture a slightly coloured product was obtained of isoxazolyl-CH, 2.25

good purity according to TLC. IR- and PMR-spectra. Analysis of the PMR spectrum of the product, 6-{[ 3-methyl-isoxazol-5-yl]acetamido l penicillanic acid,

Partial analysis of the lR-spectrum of the final product (KBr, values in cm):

I 780 C =O/3-lactam I750 C=O ester I670 C=O amide I230 COC ester I380 and I420 isoxazole ring absorptions EXAMPLE XXIII Sodium salt of 6-{a-(N-phenyUcarbamyI-l3-(2.6-dichloro)phenylisoxazol-S-yllacetamidoipenicillanic acid.

A solution of 1.0 g (2.9 mmol) of N-phenyl-3-(2.6 dichloro) phenyl-isoxazol-S-yI-acetamido in 15 ml of dry tetrahydrofurane was cooled down to 70 C. At 70 C were in succession dropwise introduced a precooled solution of about 2.9 mmol of n-butyllithium in 5 ml of a mixture of n-hexane and dry tetrahydrofurane, next 0.44 ml (about 2.9 mmol) of N,N,N.N'- tetramethylethylene-diamine and finally again a precooled solution of 2.9 mmol of n-butyllithium in 5 ml of a mixture of n-hexane and dry tetrahydrofurane. The reaction mixture was additionally stirred during 1 hour at 70 C. To the in this manner prepared reagent was subsequently added dropwise (at 70 C) a solution of 0.91 g (2.9 mmol) of trimethylsilyl 6- isocyanatopenicillanate in 5 ml of dry toluene. After completion of the addition the temperature of the reaction mixture was allowed to rise to 50 C. at which temperature stirring was continued during approximately 30 minutes. Then, the reaction mixutre and diluted hydrochloric acid were added simultaneously to a well stirred mixture of 30 ml of water and 30 ml of diethylether cooled down to 0 C. The rates of addition were mutually balanced to give a pH of approximately 7.5 throughout the neutralisation. The resulting layers were separated and the water-layer for purification once extracted with 30 ml of diethyl ether and once with 30 ml of ethyl acetate. The combined organic layers and the water-layer were inspected by thin-layer chromatography (detection of sulphur containing compounds) with as eluent a 98:2 mixture of diethylether and formic acid. The combined organic layer did not contain such compounds and was discarded. The chromatogram of the water-layer showed four wellseparated spots, three minor ones and one major spot. The minor spots were attributed respectively to degradation product(s). to N,N'-dipenicillanylurea and to n-butyI-carbonamido-penicillanic acid. The Rf-values of the latter two spots were found to be equal to the Rfvalues of the actual peniciilins. In order to separate the compound responsible for the fourth and major spot on the chromatogram, the water-layer was extracted at pH 4.9 and pH 3.6 with 30 ml of diethyl ether, which resulted in complete removal of the desired compound from the water-layer. The remainder and part of the third compound (presumably n-butylpenicillin) were removed by extraction at pH 3.3 with a 2:1 mixture of diethyl ether and ethylacetate. In order to remove the byproduct, this layer was repeatedly washed with iced water of pH 4.6, which resulted in another (the third) almost clean extract and a number of washings still containing considerable amounts of the desired product. The fourth extract was obtained by extraction of the combined washings at pH 6.0 with ethyl acetate. The four extracts were combined, washed with iced water, dried on anhydrous magnesium sulfate, filtered and concentrated in vacuo. The concentrated solution of the desired compound in ethyl acetate was treated with a concentrated solution of sodium a-ethyl capronate in ethyl acetate. The sodium salt of the penicillin was precipitated from this addition of dry diethyl ether. The precipitate was collected by filtration, washed with diethyl ether and dried to constant weight, to obtain 580 mg of the sodium salt of 6-{a-(N-phenyl)carbamyI-[3- (2.6-dichloro)phenyl-isoxazol-S-yl] acetamido penicillanic acid. The final product was inspected by thinlayer chromatography, IR spectra and PMR spectra, which confirmed the alleged structure and indicated the impurities of the final product: some sodium a-ethyl capronate and a slight amount of degradation product(s).

Partial analysis of the IR spectra of the final product and of the starting product (solutions in chloroform. conc. about IO rug/ml, values in cm):

Final Product N-phenyl-3-( 2 .(a-dichloro) amldo NH (presumably of C ,H 3430 NH NHCO) NH (broad) 1695 C=O amide (intensive) I598 C=C arom. (intensive) I557 C% and/or C=N (sharp. medium intensity) Continued 1500-1550 presumably NH dcformu- :1520 presumably NH defortion mation (medium intensity) 1495 C=C arom. [medium in 1496 alrom. (medium intens.) tens.) :1-140 mcd.intens. isoxazolcring} I435 intensive }isoxuzolering I380 mcd.intcns. absorptions 1380 medjntens. absorptions 783 (in KBr disc) (-Cl lintens.) 785 (in KBr disc] C-Cl medium I 772 (in KBr disc) intensities 752 (in KBr disc) possibly uroma- 755 (in KBr disc) possibly tic suhst. pat. [med aromatic suhst.pat. (med.

intensity) intensity) EXAMPLE XXIV (H -CO about-1.0 (slightly broadened singlet. 2

protons) S-CH about 3.6 (center of AB- uartet with \er weak 7-{l3-(4-mtro)phenyl-isoxaaol-5 yllacetamldolcephouter mi 2 Protons) q i alosporamc acid. C0-CH,. 2.06 (3 protons) Using the procedure of Example 111 3-(4-nitro)phenyl-isoxazol-S-yl acetyl chloride (prepared from 1.64 g. (6.6 mmol) of the corresponding acetic acid and thionyl chloride) was brought into reaction with N.O-biscording to thin-layer chromatography. 1R and PMR spectra. the purity of the final product, 7-{[3-(4- nitro)phenyl-isoxazol-5-yl]acetamido}cephalosporanic acid, was about 85 Analysis of the PMR spectrum of the final product dissolved in hexadeuterodimethylsulphoxide (60 Mc, fi-values in ppm. internal reference 2.Z-dimethylsilapentane-S-su1phonate):

EXAMPLE XXV The compounds of examples 1 to XX were tested for antibiotic activity in vitro with an agar serial dilution test which was carried out as follows: A stock of the antibiotic at 2.000 g/ml was prepared in a sterile suitable vehicle and two-fold dilutions were made with sterile l/20 M01 phosphate buffer pH 6.5 (KH PO NaOH). 1 ml quantities of each dilution were incorporated in 19 m1 brain-heart infusion agar in sterile Petri dishes and the hardened surface was inoculated with test organisms and incubated 24 hours at 37 C. The minimal in- N-H 9.36 and 9.22 (.l=8.0 10.5 cps. about 0.8

proton] cm 7.9 q 85 (AA.BB.sp|ming Paucm 4 hlbitory concentration (MIC) was expressed in ug/ml. protons) the least amount of antibiotic that completely inhibited isoxazolyl C.H 7.05 1 proton) c, i-i 5.37. 5.79. 5.73 and 5.65 (slightly broadened the test organism. The MIC values of the product, and

signals. .I 8.0 cps and J 4.6 cps. 1 proton) CW 5J9 and in UM :46 :01 cps of Cloxazzllme, Nafcillm, Dicloxacillin, Cephalexm,

h inadC halridi,arfr 04:? (519) 4.97 4.82 and 460 3 protons Cep alot ep 0 n s e e ences are shown (1 13.0 1 0.2 cps) 1n the following tables:

MlC's in ,ug/ml Test Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Organism 1 ll 11! 1V V VI VII VIII lX X Gram os. Bacillus subtilis ATCC 6633 0.007 0.007 10.007 1 0.5 0.03 0.015 0.25 0. 12 0.005 Staphylococcus aurel s A 0.01 0.01 0.015 0.75 l 0.06 0.06 1.5 0.25 0.03 A321 0.015 0.015 20.007 0.5 1 0.06 0.06 0.5 0.25 0.03 A355) 3 3 0.12 3 12. 3 0.25 3 25 3 L 1601)) 1 1 0.06 1.5 3 0.5 0.12 3 25 0.5 Streptococcus huemolyticus A266 0.007 0.007 10.007 0.5 0.5 0.012 0.03 0.25 0.06 0.006 Streptococcus fuecalis L 50 50 0.5 12.5 25 0.25 l 1.5 0.25 Diplococcus pneumoniuc L 54 0.25 0.25 +0007 0.75 6 0.03 0.03 0.12 0.02 Gram neg. Brucella melitensis A488 0.5 0.5 1 12.5 25 3 12.5 12.5 25 3 Pasteurellu multocidu A723 2 2 l 6 1.5 12.5 50 100 25 3 Klebsiella pneumoniue A809 100 100 12.5 100 100 100 50 100 100 100 '1 Ponielllinule-nrnducing MlCs in ug/ml Test Example Example Example Example Example Example Example organism XI Xll Xlll XIV XV XVI XVII Gram os. Bacillus Subtilis ATCC 6633 0.06 0.06 l 3 0.25 0.03 0.5 Staphylococcus aureus A55 0.12 0.25 3 12.. 0.5 0.5 l

A32l 0.06 0.12 1.5 12.5 I 0.03 1

L160a') 12.5 0.25 6 100 12.5 12.5 6 Streptococcus haemolyticus A266 0.007 0.06 0.5 1.5 0.06 0.015 0.06 Streptococcus faecalis L 80 1.5 6 I 50 0.5 0.25 3 Diplococcus pneumoniae L 54 (H2 006 1.5 6 0.03 0.06 0.5 Gram neg. Brucella melitensis A488 3 511 100 50 6 3 25 Pasteurella multocida A723 50 100 100 50 12.5 6 50 Klebsiella pneumoniae A809 100 100 100 100 100 100 100 MlCs in #g/ml Test Example Example Example Cloxa- Naf- Dicloxa- Cephale- Cepha- Cephaloorganism XVIII XIX XX zilline cilline cilline xine lotine ridine Gram pos. Bacillus subtilis ATCC 6633 0.06 0.(J 0.25 0.25 0.5 0. I 2 0.5 0.03 0.06 Staphylococcus A55 0.25 0.03 0.25 0.12 0.12 0.06 3 0.25 0.06

A355) 12.5 I 3 0.5 I 0.5 12.5 I 012 L160) 12.5 3 3 I 0.5 0.25 12.5 I 0.06 Streptococcus haemolyticus A266 0.015 1.5 0.03 0.25 0.015 0.06 0.25 0.06 0.015 Streptococcus faecalis L 80 l l 0.5 10 12.5 100 25 12.5 Diplococcus pneumoniae L 54 0.12 0.03 0.06 1.5 0.06 0.5 3 0.25 0.015 Gram neg. Brucella melitensis A488 0.5 0.12 3 100 6 100 6 6 3 Pasteurella multocida A723 6 I 6 6 l2. 6 3 0.25 I Klehsiella pncumoniac A809 100 100 100 6 I2. 25 3 0.5 3

MlC's il'l #g/ml EXAMPLE XXVI Q T QQ A. The compounds prepared in examples I, ll, III, IV, V. VII, IX and XI were tested in vivo together with Gram pos. some reference compounds.

Bacillus subtilis ATCC 6633 0.06 0.06 s'aphyhwccus 22: 8'2 Tested animals: female mice (Swiss), weight 20 g.

i 40 Infection way: intraperitoneal 1 1 3 Therapeutics: the tested compound dissolved in a sl'epwcoccus g'g 0 12 (m3 physiological NaCl-solution, 3 X l/3 close after Streptococcus Faecalis L 25 3 every 2 hours. The first dose was administered just Diplococcus L54 0.5 1.5 after h i f i pneumoniae Brucella melitensis A488 12.5 3 45 Pflsteurella multocidfl A723 3 ED calculation: accordin to H 19 6 Klebsiella pneumoniae A809 3 g om 5 The results are summarized in the following table.

ED.-,..mg/kg in experiments with mice.

Example 1111 Example V Example III Inf. i.p. i.p. i.p. Ther. i.p. s.c. p.o. i.p. s.c. p.o. i.p. s.c. p.o.

0.465 B 215 215 215 2.87 215 :200 C 133.0 215 215 1.78 75.0 215 D 213 z 200 E 215 215 p 300 300 z 200 Example IV Example VII Example IX Example XI Inf. i.p. i.p. i.p. i.p.

Ther. i.p. s.c. p.o. i.p. s.c. p.o. i.p. s.c. p.o. i.p. s.c. p.o.

A 23.3 31.6 50.1 3.8 18.5 70.0 2 l s 100 21.5 100 B 23.3 215 :200 17.1 140.0 215 Continued Example lV Example Vll Example IX Example XI Thor. i.p. sc. p.o. irp. s.c. p.o. i.p. s.ci p.o. i.p. s.c. p.o.

E Z| 2l5 2l5 300 F 2l5 2l5 :215 300 300 Propicillin Dicloxacillin Keflin (Cefulo in) Inf. i.pi i.p, i.p.

Ther. i.p. s.c. p.o. i.p. s.c. p.o i.p. sc. p.o.

A 0.926 2.33 10.8 0.909 126 92.6 H90 162 l4.7 B 133 2l5 215 l.7l 163 ll[) 27.] Zl5 68.| C 88 2l5 ZlS 5.84 92.6 J16 20 HO l4? D 79.4 I 200 E 2l5 215 F 79.4 68.l

A Staphylococcus nureus A .Zl

B Staphylococcus nurcus A 2001 C Staphylococcus :lurcus A 2001! D Salmonella typhi murium R I72 E Pseudomonas acruginosn A 1058 F Proteus mirahilis (1T.

miceinfection admini- ED, in mg/kg Dicloxagroup way stration Tested compound cillin A i.p. itp. 21.5-46.5 2l.5

i.p. per as I 465 46.5400

B i.p. i.p. 2|.5 46.5-100 i.p. per os 2l.5 215-465 C. Serum levels of the tested compound and dicloxacillin. The serum levels of dicloxacillin and the compound of example III were determined after an intramuscular administration of 50 mg/kg of these compounds in an aqueous solution in rabbits. The serum levels are reported in the following table.

Hours after Serum level Test compound injection in I'lml Dicloxacillin 1 2 2 l8.8 4 7! Example H] l 5.4 4 2.2

Peak blood levels of the two drugs were reached after l hour with the tested compound giving levels of 5.4 lg/ml and dicloxacillin 27 ug/ml. After 4 hours. these levels were 2.2 pig/ml and 7.1 ag/ml respectively. Dicloxacillin was almost completely bound to serum protein whereas the tested compound appeared to be bound to the extent of about 50%. Thus, the amount of free drug in the serum was of the same order. However, the M.I.C. values for the tested compound were approximately 5 l0 times less so that the overall result was better and this was reflected in the in vivo results.

EXAMPLE XXV" EXAMPLE XXVIII Syrups were prepared from the compounds obtained according to Examples l-XXIV by mixing the following ingredients:

sodium salt of the desired compound L5 6 g soluble starch l 3 g sodium saccharin 0.] l g nipa M 0.06 g strawberry flavor 0.! 5 g amaranth 0.010 g saccharose 30 g water added to a volume of (10 ml These prepared syrups were suitable for oral administration.

EXAMPLE XXIX Capsules were prepared in the usual way containing as active ingredient the compound obtained according to Examples I-XXIV. The components of the capsules are listed below:

sodium salt of the desired compound l50500 mg potassium bicarbonate -300 mg magnesium stearate 2- l0 mgv lactose q.s. for 1 capsule These capsules could be used for oral administration.

EXAMPLE XXX Tablets were prepared in the usual way containing as active ingredient the compounds of Examples l XXIV. The components of the tablets are listed below:

sodium salt of the desired compound l25-500 mg polyvinylpyrrolidine S- 30 m amylum maidis 100-300 mg magnesium stearate l- 20 m lactose q.s. for 1 tablet 

1. A COMPOUND OF THE FORMULA
 2. A compound of claim 1 wherein R is selected from the group consisting of 2,6-dichlorophenyl and 2-chloro-6-fluorophenyl, R1 and R2 are selected from thE group consisting of hydrogen and methyl, Q is a cephalosporanic acid nucleus and pharmaceutically acceptable salts of said acids.
 3. A compound of claim 1 selected from the group consisting of 7-((3-(2,6-dichlorophenyl)-isoxazol-5-yl)acetamido)cephalosporanic acid and its non-toxic, pharmaceutically acceptable salts.
 4. A compound of claim 1 selected from the group consisting of 7-((3-(2,6-dichlorophenyl)isoxazol-5-yl)acetamido)desacetoxycephalosporanic acid and its non-toxic, pharmaceutically acceptable salts.
 5. A compound of claim 1 selected from the group consisting of 7-((3-(2,4,6-trimethyl)phenyl-isoxazol-5-yl) acetamido)cephalosporanic acid and its non-toxic, pharmaceutically acceptable salts.
 6. A compound of claim 1 selected from the group consisting of 7-((3-(2,6-dichloro)phenyl-4-methyl-isoxazol-5 -yl)acetamido)desacetoxycephalosporanic acid and its non-toxic, pharmaceutically acceptable salts.
 7. A compound of claim 1 selected from the group consisting of 7-((3-(2,4,6-trimethyl)phenyl-4-methyl-isoxazol-5 -yl)acetamido)cephalosporanic acid and its non-toxic pharmaceutically acceptable salts.
 8. A compound of claim 1 selected from the group consisting of 7-((3-(2,4,6-trimethyl)phenyl-4-methyl-isoxazol-5-yl)acetamido)desacetoxy -cephalosporanic acid and its non-toxic, pharmaceutically acceptable salts.
 9. A compound of claim 1 selected from the group consisting of 7-((3-(4-nitro)phenyl-isoxazol-5-yl)acetamido) cephalosporanic acid and its non-toxic, pharmaceutically acceptable salts.
 10. A compound of claim 1 wherein R is selected from the group consisting of 2,6-dichlorophenyl and 2,4,6-trimethylphenyl, R1 is selected from the group consisting of hydrogen, chlorine, and lower alkyl and R2 is selected from the group consisting of hydrogen, chlorine, amino and lower alkyl. 